Insights into Adaptive Regulation of the Leaf-Petiole System: Strategies for Survival of Water Lily Plants under Salt Stress

The water lily (Nymphaea tetragona) is an ancient angiosperm that belongs to the Nymphaeaceae family. As a rooted floating-leaf plant, water lilies are generally cultivated in fresh water, therefore, little is known about their survival strategies under salt stress. Long-term salt stress causes morphological changes, such as the rapid regeneration of floating leaves and a significant decrease in leaf number and surface area. We demonstrate that salt stress induces toxicity soon after treatment, but plants can adapt by regenerating floating leaves that are photosynthetically active. Transcriptome profiling revealed that ion binding was one of the most-enriched GO terms in leaf-petiole systems under salt stress. Sodium-transporter-related genes were downregulated, whereas K+ transporter genes were both up- and downregulated. These results suggest that restricting intracellular Na+ importing while maintaining balanced K+ homeostasis is an adaptive strategy for tolerating long-term salt stress. ICP-MS analysis identified the petioles and leaves as Na-hyperaccumulators, with a maximum content of over 80 g kg−1 DW under salt stress. Mapping of the Na-hyperaccumulation trait onto the phylogenetic relationships revealed that water lily plants might have a long evolutionary history from ancient marine plants, or may have undergone historical ecological events from salt to fresh water. Ammonium transporter genes involved in nitrogen metabolism were downregulated, whereas NO3−-related transporters were upregulated in both the leaves and petioles, suggesting a selective bias toward NO3− uptake under salt stress. The morphological changes we observed may be due to the reduced expression of genes related to auxin signal transduction. In conclusion, the floating leaves and submerged petioles of the water lily use a series of adaptive strategies to survive salt stress. These include the absorption and transport of ions and nutrients from the surrounding environments, and the ability to hyperaccumulate Na+. These adaptations may serve as the physiological basis for salt tolerance in water lily plants.


Introduction
The water lily is a perennial floating plant of the Nymphaeaceae family, which is one of the most ancient angiosperm lineages [1]. Water lilies are famous for their colorful flowers and strong fragrance [2,3]. The water lily flower is also widely used for essential oil extraction and its medicinal properties [4,5]. Most studies of the water lily mainly focus on genetic evolution, flowering rhythm, essential oil extraction, and seed biology. However, very little information exists about the mechanism of water lily salt tolerance in saline waters [1,3,5,6]. Water lilies are generally cultivated in fresh water, which may explain why so few studies have investigated their responses to salt stress. According to a vegetation survey conducted in the northern part of the southeast island of Scotland, the

Phenotypes of Water Lily Plants under Salt Stress
Nymphaea 'Colorado' water lily plants were treated with 0, 50, 100, 150, and 200 mM NaCl for 30 days. Symptoms of salt stress appeared in plants treated with 50 mM NaCl with severity increasing as the concentration of NaCl increased. Salt stress greatly inhibited the growth of water lily plants. The number of floating leaves decreased, with leaf chlorosis and decay after salt treatment. The images demonstrate a rapid reduction in biomass and the number of floating leaves in plants treated with as little as 50 mM NaCl. Decaying mature leaves and the regeneration of young floating leaves are clearly seen in plants treated with NaCl after 30 days of treatment ( Figure 1). Leaf surface area, leaf number, and plant biomass were all negatively correlated with the concentration of NaCl after 30 days of continuous treatment ( Figure 2). Quantification of the average leaf area, total leaf area, and average number of leaves confirmed that these parameters declined as the salt concentration increased (Figure 2a-c).
With reductions in leaf area and leaf number likely limiting the photosynthetic capacity of salt-treated water lilies, we anticipated reductions in plant fresh weight (FW) and dry weight (DW). The results confirmed that the FW and DW of petioles and floating leaves were also negatively correlated with the severity of salt stress (Figure 2d). The effects were apparent in as little as 50 mM NaCl with reductions in FW and DW of 44.82% and 36.75%, respectively, With reductions in leaf area and leaf number likely limiting the photosynthetic capacity of salt-treated water lilies, we anticipated reductions in plant fresh weight (FW) and dry weight (DW). The results confirmed that the FW and DW of petioles and floating leaves were also negatively correlated with the severity of salt stress (Figure 2d). The effects were apparent in as little as 50 mM NaCl with reductions in FW and DW of 44.82% and 36.75%, respectively, relative to the control plants. Plants treated with 200 mM NaCl exhibited 94.75% and 96.20% reductions in FW and DW, respectively, compared with the control plants.

Effects of Salt Stress on Photosynthesis in Water Lily Plants
Multiple photosynthetic parameters were recorded at three-day intervals over a 30day period in plants treated with 0, 50, 100, 150, and 200 mM NaCl. A rapid decline in the leaf net photosynthetic rate (Pn) occurred by day 3 for all treatments, and the decline was greater with increasing salt concentration (Figure 3a). After 30 days, the 50 and 100 mM NaCl treatments resulted in a Pn decrease of 17.69% and 49.49%, respectively, relative to the control. At higher concentrations of 150 and 200 mM NaCl, Pn decreased by 62.01% and 82.62%, respectively, relative to the negative control. Notably, the Pn stabilized after  With reductions in leaf area and leaf number likely limiting the photosynth pacity of salt-treated water lilies, we anticipated reductions in plant fresh weig and dry weight (DW). The results confirmed that the FW and DW of petioles and leaves were also negatively correlated with the severity of salt stress (Figure 2d) fects were apparent in as little as 50 mM NaCl with reductions in FW and DW of and 36.75%, respectively, relative to the control plants. Plants treated with 200 m exhibited 94.75% and 96.20% reductions in FW and DW, respectively, compared w control plants.

Effects of Salt Stress on Photosynthesis in Water Lily Plants
Multiple photosynthetic parameters were recorded at three-day intervals ov day period in plants treated with 0, 50, 100, 150, and 200 mM NaCl. A rapid declin leaf net photosynthetic rate (Pn) occurred by day 3 for all treatments, and the dec greater with increasing salt concentration (Figure 3a). After 30 days, the 50 and NaCl treatments resulted in a Pn decrease of 17.69% and 49.49%, respectively, re the control. At higher concentrations of 150 and 200 mM NaCl, Pn decreased by and 82.62%, respectively, relative to the negative control. Notably, the Pn stabiliz day 6 for plants treated with 50 mM NaCl. This stabilization effect was also obs higher salt concentrations but occurred after 12 to 15 days, and the stabilized Pn were reduced by approximately 50% or more compared to the negative contr

Effects of Salt Stress on Photosynthesis in Water Lily Plants
Multiple photosynthetic parameters were recorded at three-day intervals over a 30-day period in plants treated with 0, 50, 100, 150, and 200 mM NaCl. A rapid decline in the leaf net photosynthetic rate (Pn) occurred by day 3 for all treatments, and the decline was greater with increasing salt concentration (Figure 3a). After 30 days, the 50 and 100 mM NaCl treatments resulted in a Pn decrease of 17.69% and 49.49%, respectively, relative to the control. At higher concentrations of 150 and 200 mM NaCl, Pn decreased by 62.01% and 82.62%, respectively, relative to the negative control. Notably, the Pn stabilized after day 6 for plants treated with 50 mM NaCl. This stabilization effect was also observed at higher salt concentrations but occurred after 12 to 15 days, and the stabilized Pn values were reduced by approximately 50% or more compared to the negative control. This stabilization in the Pn suggests that water lilies can tolerate photosynthetic damages caused by salt stress, and indicates that increased photosynthetic damage likely occurs when plants are subjected to salt concentrations greater than 50 mM NaCl. ative stability in the time after salt treatment ( Figure 3b). However, no significant changes were observed in the transpiration rate (Tr) or stomatal conductance (Gs) between treatments at each time point (Figure 3c,d). As a typical floating plant, water lilies have no stomata in their lower epidermis, so stomatal openings are only distributed in the upper epidermis of mature leaves ( Figure S1). Therefore, we speculate that the decline in Pn induced by salt stress depended mainly on non-stomatal factors. To further determine the effects of salt stress on water lily plants, chlorophyll fluorescence parameters of floating leaves were measured. A rapid decline in the photosystem II (PSII) actual photochemical quantum yield (ΦPSII), electron transport rate (ETR), and photochemical quenching coefficient (qP) occurred within the first nine days after salt stress treatment (Figure 4b-d). These results indicated that salt stress may decrease the rate of photochemical reactions in the floating leaves, and the energy absorbed by chlorophyll in PSII may dissipate through heat and fluorescence. In leaves treated with 50 and 100 mM NaCl, a near-complete recovery of chlorophyll fluorescence parameters was achieved after 18 days. This result suggests that the chlorophyll in water lily leaves can eventually acclimate to NaCl concentrations of 100 mM or less. At NaCl concentrations higher than 150 mM, water lily plants could survive, maintain continuous growth, and regenerate young floating leaves but suffer from more severe and prolonged effects on photosynthesis. The intercellular CO 2 concentration (Ci) increased initially, but was followed by relative stability in the time after salt treatment ( Figure 3b). However, no significant changes were observed in the transpiration rate (Tr) or stomatal conductance (Gs) between treatments at each time point (Figure 3c,d). As a typical floating plant, water lilies have no stomata in their lower epidermis, so stomatal openings are only distributed in the upper epidermis of mature leaves ( Figure S1). Therefore, we speculate that the decline in Pn induced by salt stress depended mainly on non-stomatal factors.
To further determine the effects of salt stress on water lily plants, chlorophyll fluorescence parameters of floating leaves were measured. A rapid decline in the photosystem II (PSII) actual photochemical quantum yield (Φ PSII ), electron transport rate (ETR), and photochemical quenching coefficient (qP) occurred within the first nine days after salt stress treatment (Figure 4b-d). These results indicated that salt stress may decrease the rate of photochemical reactions in the floating leaves, and the energy absorbed by chlorophyll in PSII may dissipate through heat and fluorescence. In leaves treated with 50 and 100 mM NaCl, a near-complete recovery of chlorophyll fluorescence parameters was achieved after 18 days. This result suggests that the chlorophyll in water lily leaves can eventually acclimate to NaCl concentrations of 100 mM or less. At NaCl concentrations higher than 150 mM, water lily plants could survive, maintain continuous growth, and regenerate young floating leaves but suffer from more severe and prolonged effects on photosynthesis.

RNA-Seq Analysis of Leaves and Petioles from Water Lily Plants Treated with NaCl
We used the Illumina NovaSeq 6000 system to perform RNA-seq on young floating leaves and petioles from N. 'Colorado' water lily plants treated with 150 mM NaCl. The goal of this experiment was to investigate the gene networks under long-term salt stress acclimation in water lilies. The sequenced cDNA libraries generated between 20.4 and 24.0 million raw reads. The total number of clean reads per library ranged from 19.8 to 23.7 million after removing poly-N reads, adapters, and low-quality reads ( Table 1). The length distributions of unigenes are available in Figure S2. The clean reads were assembled into transcripts using Trinity software (v2.4.0). The transcripts were mapped to the NCBI RefSeq database, and the mapping rate range from 66.72% to 69.92% (Table S1).
The sequences were compared to the Nr, Nt, Pfam, KOG/COG, Swiss-Prot, KO, and GO database and 136,306 genes were annotated (Table S2). FPKM values were calculated to quantify the differentially expressed unigenes. The FPKM density distribution is shown in Figure S3. Pearson's correlation coefficient showed a relatively high correlation between biological replicates of the plant samples ( Figure S4).

RNA-Seq Analysis of Leaves and Petioles from Water Lily Plants Treated with NaCl
We used the Illumina NovaSeq 6000 system to perform RNA-seq on young floating leaves and petioles from N. 'Colorado' water lily plants treated with 150 mM NaCl. Th goal of this experiment was to investigate the gene networks under long-term salt stres acclimation in water lilies. The sequenced cDNA libraries generated between 20.4 and 24. million raw reads. The total number of clean reads per library ranged from 19.8 to 23. million after removing poly-N reads, adapters, and low-quality reads ( Table 1). The length distributions of unigenes are available in Figure S2. The clean reads were assembled into transcripts using Trinity software (v2.4.0). The transcripts were mapped to the NCBI Ref Seq database, and the mapping rate range from 66.72% to 69.92% (Table S1). The se quences were compared to the Nr, Nt, Pfam, KOG/COG, Swiss-Prot, KO, and GO data base and 136,306 genes were annotated (Table S2). FPKM values were calculated to quan tify the differentially expressed unigenes. The FPKM density distribution is shown in Fig  ure S3. Pearson's correlation coefficient showed a relatively high correlation between bi ological replicates of the plant samples ( Figure S4).

Analysis of Differentially Expressed Genes
Differentially expressed genes (DEGs) were identified using DEseq by comparing the transcriptomes of the leaves and petioles collected from salt-treated plants to a nontreated control. A total of 2292 and 2355 DEGs were identified in the leaves and petioles, respectively, of water lilies treated with 150 mM NaCl. Of the DEGs, 296 were upregulated in young floating leaves, and 1996 were downregulated. In the petioles, 476 genes were upregulated, and 1879 were downregulated ( Figure 5a). Floating leaves and petioles subjected to 150 mM NaCl shared 545 DEGs, whereas 1747 DEGs were leaf-specific, and 1810 DEGs were petiole-specific ( Figure 5b). of control plants; CL, leaves of control plants; SP, petioles of salt-treated plants; SL, leaves treated plants.

Analysis of Differentially Expressed Genes
Differentially expressed genes (DEGs) were identified using DEseq by compar transcriptomes of the leaves and petioles collected from salt-treated plants to treated control. A total of 2292 and 2355 DEGs were identified in the leaves and p respectively, of water lilies treated with 150 mM NaCl. Of the DEGs, 296 were upreg in young floating leaves, and 1996 were downregulated. In the petioles, 476 gene upregulated, and 1879 were downregulated ( Figure 5a). Floating leaves and petiol jected to 150 mM NaCl shared 545 DEGs, whereas 1747 DEGs were leaf-specific, an DEGs were petiole-specific ( Figure 5b).

Significantly Enriched Pathways in Water Lilies under Salt Stress
Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG yses were performed to identify significantly enriched pathways from the DEGs. were categorized using Goseq software with Wallenius' noncentral hypergeomet tribution, and the GO terms with corrected p-values less than 0.05 were considered icantly enriched. Twenty GO terms were identified as significantly enriched with from floating leaves, and eleven GO terms were identified as significantly enriche DEGs from petioles (Figure 6a,b). Notably, the ion binding pathway (GO:004316 significantly enriched in both the leaves and petioles: 443 DEGs in leaves and 396 in petioles. This result suggests that ion binding in the leaves and petioles is an imp biological process used by water lilies for adaptation to salt stress.

Significantly Enriched Pathways in Water Lilies under Salt Stress
Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed to identify significantly enriched pathways from the DEGs. Genes were categorized using Goseq software with Wallenius' noncentral hypergeometric distribution, and the GO terms with corrected p-values less than 0.05 were considered significantly enriched. Twenty GO terms were identified as significantly enriched with DEGs from floating leaves, and eleven GO terms were identified as significantly enriched with DEGs from petioles ( Figure 6a,b). Notably, the ion binding pathway (GO:0043167) was significantly enriched in both the leaves and petioles: 443 DEGs in leaves and 396 DEGs in petioles. This result suggests that ion binding in the leaves and petioles is an important biological process used by water lilies for adaptation to salt stress. Notable pathways enriched in both leaves and petioles from the KEGG analysis included starch and sucrose metabolism and plant hormone signal transduction ( Figure  7a,b). In total, 36 genes involved in starch and sucrose metabolism were downregulated under salt stress. This decrease in carbohydrate metabolism might explain the reduced photosynthetic capacity of plants subjected to salt stress. Genes involved in plant hormone signal transduction included DEGs related to auxin, ethylene, abscisic acid, and gibberellin that were identified in water lily plants under salt stress, and auxin-related genes accounted for more than 50% DEGs. Notable pathways enriched in both leaves and petioles from the KEGG analysis included starch and sucrose metabolism and plant hormone signal transduction (Figure 7a,b). In total, 36 genes involved in starch and sucrose metabolism were downregulated under salt stress. This decrease in carbohydrate metabolism might explain the reduced photosyn-thetic capacity of plants subjected to salt stress. Genes involved in plant hormone signal transduction included DEGs related to auxin, ethylene, abscisic acid, and gibberellin that were identified in water lily plants under salt stress, and auxin-related genes accounted for more than 50% DEGs. Notable pathways enriched in both leaves and petioles from the KEGG analysis included starch and sucrose metabolism and plant hormone signal transduction ( Figure  7a,b). In total, 36 genes involved in starch and sucrose metabolism were downregulated under salt stress. This decrease in carbohydrate metabolism might explain the reduced photosynthetic capacity of plants subjected to salt stress. Genes involved in plant hormone signal transduction included DEGs related to auxin, ethylene, abscisic acid, and gibberellin that were identified in water lily plants under salt stress, and auxin-related genes accounted for more than 50% DEGs.

Ion and Water-Transport-Related Genes in Water lily Plants under Salt Stress
The GO and KEGG analyses highlighted the importance of ion binding in the response to salt stress in water lily leaves and petioles. We decided to take a closer look at DEGs involved in ion absorption and ion transport. A total of 65 DEGs involved in ion transport were identified in both the leaves and petioles. These DEGs included genes encoding sodium transporters, potassium transporters, chloride channels, anion channels, cation transporters, and ABC transporters. Thirty-eight of these DEGs were differentially expressed in the leaves, thirty-four in the petioles, and only six overlapped in both leaves and petioles. Thirty-seven DEGs were downregulated under salt stress. Two differentially expressed sodium transporter genes (Cluster-3509.15636 and Cluster-3509.89148) were downregulated in water lily plants under salt stress. Gene Cluster-3509.15636 was

Ion and Water-Transport-Related Genes in Water lily Plants under Salt Stress
The GO and KEGG analyses highlighted the importance of ion binding in the response to salt stress in water lily leaves and petioles. We decided to take a closer look at DEGs involved in ion absorption and ion transport. A total of 65 DEGs involved in ion transport were identified in both the leaves and petioles. These DEGs included genes encoding sodium transporters, potassium transporters, chloride channels, anion channels, cation transporters, and ABC transporters. Thirty-eight of these DEGs were differentially expressed in the leaves, thirty-four in the petioles, and only six overlapped in both leaves and petioles. Thirty-seven DEGs were downregulated under salt stress. Two differentially expressed sodium transporter genes (Cluster-3509.15636 and Cluster-3509.89148) were downregulated in water lily plants under salt stress. Gene Cluster-3509.15636 was downregulated in both the leaves and petioles, whereas gene Cluster-3509.89148 was specifically downregulated in the petioles (Table 2). Based on these expression data, it seems that water lilies may adapt to salt stress by shutting down sodium transport to restrict excessive sodium from accumulating in the plant. As it was reported that AQPs can mediate ion transport across the membranes, we further identified water-transport-related DEGs in water lily plants. In total, four water-transport-related DEGs were identified in water lily plants. Notably, two aquaporin TIPs (Cluster-3509.66551 and Cluster-3509.74762) were identified as significantly downregulated genes in floating leaves ( Table 2).  Since nitrogen metabolism disorder is the main cause of salt damage in plants, we further investigated the expression of nitrogen uptake and transport-related genes. DEGs associated with nitrogen transport and metabolism were identified, including protondependent oligopeptide transporter, oligopeptide transporter, vacuolar amino acid transporter, ammonium transporter, NRT1/PTR FAMILY protein, and S-type anion channel SLAH et al. Sixteen putative transporters were differentially expressed in the leaves, seventeen in the petioles, and five in both the leaves and petioles. Genes encoding ammonium transporters were downregulated, whereas genes encoding NO 3 − transporters, including the NRT1/PTR FAMILY protein, were upregulated ( Table 2). Both upregulation and downregulation were found among the S-type SLAH family of anion channels in the leaves and petioles, suggesting a selective bias toward NO 3 − under salt stress ( Table 2).

Differential Expression of Genes Related to Plant Hormones
Plants have evolved multiple strategies for integrating exogenous salt stress signals into responses that balance growth with salt tolerance. These signals are communicated systemically through the action of different hormones, which play an important role in regulating stress responses and tolerance [22]. Auxin-related genes constitute the largest component of the plant hormone signal transduction pathway. Our RNA-seq analysis identified 19 and 24 auxin-related DEGs in the leaves and petioles, respectively ( Table 3). Most of these auxin-related genes were downregulated. The results suggested that downregulation of the auxin response pathway in salt-stressed plants may lead to reduced plant biomass. Water lilies may tolerate salt stress by inhibiting plant growth to shuttle metabolic resources toward a salt adaptation response. Ethylene is another important hormone involved in salt tolerance signaling in water lily plants. We identified five and twelve ethylene-related DEGs in the leaves and petioles, respectively. These results suggest that ethylene may act as a signaling molecule that negatively regulates salt tolerance in water lily plants. Although many reports indicate that ABA can induce adaptive responses to salt stress in maize, rice, Arabidopsis, and other species, few DEGs involved in ABA or gibberellin responses were identified in our experiment [23][24][25][26]. These results suggest that ABA and gibberellin may not play an important role in long-term salt adaptation in water lilies. To validate the expression of transcriptome data, several genes were selected for quantitative real-time PCR (qRT-PCR) ( Figure 8). Generally, the qRT-PCR results of these genes were consistent with the transcriptome data.

Changes in Sodium (Na) and Potassium (K) Contents in Salt-Stressed Leaves and Petioles
To investigate the Na and K contents in salt-stressed plants, the leaves and petiole were sampled after 150 mM NaCl treatment. The K content of leaves and petioles d creased in salt-stressed plants, with most of the decline occurring in the first three days

Changes in Sodium (Na) and Potassium (K) Contents in Salt-Stressed Leaves and Petioles
To investigate the Na and K contents in salt-stressed plants, the leaves and petioles were sampled after 150 mM NaCl treatment. The K content of leaves and petioles decreased in salt-stressed plants, with most of the decline occurring in the first three days of treatment ( Figure 9). In contrast, the Na content of leaves increased up to day 12 of treatment before stabilizing, whereas the Na content of petioles increased up to day three before stabilizing ( Figure 9). As a freshwater plant, water lilies accumulate a large amount of Na in their leaves and petioles, which results in a high Na/K ratio under salt stress. When water lilies were grown under salt stress, the petiole rapidly accumulated Na until saturation occurred around day three at a concentration of 80 g kg −1 DW. The Na content peaked within 12 days in the leaves and saturated in the petioles a few days later. Although the transcriptomic data suggest that both the leaves and petioles have an ion exchange capacity, these results highlight the important function of the petioles in Na absorption and transportation in short-term salt stress. Figure 9. Na and K contents and Na/ K ratio in water lily plants treated with 150 mM NaCl. (a content, K content, and Na/ K ratio in leaves. (b) Na content, K content, and Na/ K ratio in pet Data are the means of three biological replicates (±SDs). The asterisks represent statistically sig cant differences (p < 0.05).

Discussion
The root system of terrestrial plants is the first organ to encounter salt stress. R are also the first to induce a systemic physiological response through signal transduc ion absorption, and long-distance ion transport from the roots to the shoots [16,27]. systemic response requires coordinated gene regulation between the roots and the sh [28]. As a floating aquatic plant, water lilies have morphologically and physiologi adapted to their aquatic environment. The fact that water lily roots and shoots are surrounded by water suggests that both organs sense and respond to salt stress sim neously. In this study, we found that water lilies adapt to salt stress by decreasing number and surface area of its floating leaves along with the rapid regeneration of floating leaves (Figures 1 and 2). Photosynthetic data revealed the effects of salt-indu toxicity. A rapid drop in the photosynthetic rate and chlorophyll fluorescence param occurred soon after salt treatment. However, water lily plants exposed to lower leve salt stress were able to partially recover over time (Figures 3 and 4). These results sug that water lilies can adapt to salt stress by adjusting their morphological and physiolo strategies to become salt tolerant ( Figure 10). Water lilies have adapted to saline wat a manner that is unique and significantly different from terrestrial plants. In this st the absorption and transport of ions by the petioles and leaves are typical features by water lilies to regulate changes in environmental salt concentration. By regulating transport across the plasma membrane of stem and leaf cells, water lilies maintain ho ostasis between intracellular and extracellular salt concentrations ( Figure 10). It had reported that AQPs can mediate ion transport across the membranes [29,30]. In Arab sis, AtPIP2;1 is responsible for Na + transport, and Na + uptake and accumulation ca restricted by internalizing AtPIP2;1 from the plasma membrane [31]. AQPs are als ported as turgor sensors to regulate conductance of K + channels [32]. In water lily pl two aquaporin TIPs genes were identified as significantly downregulated genes in f ing leaves (Table 2). Possibly TIPs were involved this biological process, and work gether with sodium as osmoregulators in water lily plants.
In this study, the RNA-seq results suggest that most of the differentially expre genes were organ-specific; only 13.29% DGEs overlapped in the leaves and petioles u salt stress ( Figure 5). Further analysis of ion channels and transporters suggested tha petioles and leaves tend to utilize different ion channels and transporters under salt s ( Table 2). In addition, the KEGG pathway analysis showed that the enrichment of th bosome pathway was only found in the floating leaves under salt stress, but not in submerged petioles of water lily plants. In Arabidopsis, ribosomes are highly heterogen and each organ might need a different association of non-paralogous ribosomal prot It was reported that the expression of ribosomal protein genes varied dramatically in ferent organs [33]. In Brassica napus, it was also reported that the number of paralo Figure 9. Na and K contents and Na/ K ratio in water lily plants treated with 150 mM NaCl. (a) Na content, K content, and Na/ K ratio in leaves. (b) Na content, K content, and Na/ K ratio in petioles. Data are the means of three biological replicates (±SDs). The asterisks represent statistically significant differences (p < 0.05).

Discussion
The root system of terrestrial plants is the first organ to encounter salt stress. Roots are also the first to induce a systemic physiological response through signal transduction, ion absorption, and long-distance ion transport from the roots to the shoots [16,27]. This systemic response requires coordinated gene regulation between the roots and the shoots [28]. As a floating aquatic plant, water lilies have morphologically and physiologically adapted to their aquatic environment. The fact that water lily roots and shoots are both surrounded by water suggests that both organs sense and respond to salt stress simultaneously. In this study, we found that water lilies adapt to salt stress by decreasing the number and surface area of its floating leaves along with the rapid regeneration of new floating leaves (Figures 1 and 2). Photosynthetic data revealed the effects of salt-induced toxicity. A rapid drop in the photosynthetic rate and chlorophyll fluorescence parameters occurred soon after salt treatment. However, water lily plants exposed to lower levels of salt stress were able to partially recover over time (Figures 3 and 4). These results suggest that water lilies can adapt to salt stress by adjusting their morphological and physiological strategies to become salt tolerant ( Figure 10). Water lilies have adapted to saline water in a manner that is unique and significantly different from terrestrial plants. In this study, the absorption and transport of ions by the petioles and leaves are typical features used by water lilies to regulate changes in environmental salt concentration. By regulating ion transport across the plasma membrane of stem and leaf cells, water lilies maintain homeostasis between intracellular and extracellular salt concentrations ( Figure 10). It had been reported that AQPs can mediate ion transport across the membranes [29,30]. In Arabidopsis, AtPIP2;1 is responsible for Na + transport, and Na + uptake and accumulation can be restricted by internalizing AtPIP2;1 from the plasma membrane [31]. AQPs are also reported as turgor sensors to regulate conductance of K + channels [32]. In water lily plants, two aquaporin TIPs genes were identified as significantly downregulated genes in floating leaves (Table 2). Possibly TIPs were involved this biological process, and work together with sodium as osmoregulators in water lily plants.
In this study, the RNA-seq results suggest that most of the differentially expressed genes were organ-specific; only 13.29% DGEs overlapped in the leaves and petioles under salt stress ( Figure 5). Further analysis of ion channels and transporters suggested that the petioles and leaves tend to utilize different ion channels and transporters under salt stress ( Table 2). In addition, the KEGG pathway analysis showed that the enrichment of the ribosome pathway was only found in the floating leaves under salt stress, but not in the submerged petioles of water lily plants. In Arabidopsis, ribosomes are highly heterogenous, and each organ might need a different association of non-paralogous ribosomal proteins. It was reported that the expression of ribosomal protein genes varied dramatically in different organs [33]. In Brassica napus, it was also reported that the number of paralogues expressed for each ribosomal protein gene varied extensively with tissue types [34]. In this study, the floating leaves and the submerged petioles of water lily plants might have a distinct population of ribosomal protein gene transcripts, and the physiological state, such as salt-induced osmotic stress, might demand different amounts of ribosomes between organs. Moreover, salt treatment led to leaf chlorosis and decay in floating leaves, which might include the process of cell apoptosis. The decline in chloroplast protein synthesis was associated with loss of polyribosomes, and cell apoptosis also results in the degradation of ribosomes [35,36]. Therefore, the enrichment of the ribosome pathway in floating leaves indicates functional differences in the ways in which leaves and petioles adapt to salt stress. Plants are often subjected to Na toxicity and K deficiency simultaneously. Sodium itself is antagonistic toward K absorption by cells because Na uptake is linked to K efflux [43,44]. Maintaining intracellular K homeostasis is important for proper plant growth and salt tolerance [45]. Our results demonstrated that the potassium content in the leaves and petioles of water lilies decreased after salt stress, with most of the decline occurring within three days of treatment. After three days, the K content stabilized. It appears that K efflux occurs as an early response to salt stress before the petioles and leaves adapt to prevent further loss of potassium ( Figure 9). We hypothesized that the intracellular K concentration may be regulated by potassium-related transporters. Both upregulation (Cluster-3509.83265 and Cluster-3509.8544) and downregulation (Cluster-3509.60348 and Cluster-3509.61628) of potassium transporter genes were observed in the petioles ( Table 2). Water lilies may achieve K homeostasis in the petioles to improve salt tolerance. However, the leaves may use a different strategy to prevent K accumulation. Our results showed that three potassium transporter genes were downregulated in the leaves, suggesting that potassium channels may close under salt stress. ABC transporters are also involved in ion channel regulation under salt stress in plants [46][47][48]. In A. thaliana, a high expression of AtMRP5 led to an increase in the ratio of Na to K in seedlings by regulating K uptake in roots to counteract salt stress [49]. In this study, 27 ABC transporter genes were differentially expressed under salt stress. Most of these transporters belong to the ABCC and ABCG subfamilies, which play an important role in Na and K uptake and transport under salt stress [50][51][52]. Our results suggest that water lilies maintain intracellular K above a minimal threshold to acquire salt tolerance.
In summary, floating leaves and submerged petioles use a series of adaptive strategies to survive salt stress. The absorption and transport of ions and nutrients from the surrounding environment are part of this complex biological process. The ability to hyperaccumulate Na may be the physiological basis for salt tolerance in this aquatic plant. Sodium is an essential ion that can damage plants at high concentrations [37]. The extracellular concentration of Na directly impacts the intracellular ionic balance and cellular activities that rely on this balance [38,39]. Terrestrial plants have efficient ion transport and selective absorption mechanisms to maintain intracellular homeostasis. Common strategies to avoid cytotoxic levels of Na include selective absorption, efflux, and sequestration of Na [40]. It was previously reported that angiosperm species can be classified into Na-excluders, Na-responders, and Na-accumulators. Species of Caryophyllales raised in environments lacking salt with a shoot Na concentration over 4 g kg −1 DW were classified as Na hyperaccumulator species [41]. Coastal plant species with a leaf Na concentration over 30 g kg −1 DW were defined as Na hyperaccumulators [42]. Upon salt treatment, Na hyperaccumulation occurred in the leaves and petioles of water lilies within 3 days. While they are generally considered to be freshwater plants, our results suggest water lilies exhibit the characteristics of Na hyperaccumulators. In the absence of salt, the Na content in floating leaves exceeded 15 g kg −1 DW, which is higher than that of many terrestrial plants. The Na content in the petioles of water lilies exceeded 40 g kg −1 DW, which is higher than that of coastal plants that hyperaccumulate Na. These results suggest that water lily plants naturally hyperaccumulate Na, especially in the petioles. Under salt stress, hyperaccumulation of Na occurred in both the petioles and leaves of water lilies. Three days after the salt treatment commenced, the Na content in the petioles was nearly 80 g kg −1 DW, which is almost two-fold greater than that in the petioles of plants raised in fresh water. The Na content of the leaves also rose to 80 g kg −1 DW after 12 days of treatment, which was nearly five-fold greater than that in the leaves of plants raised in fresh water (Figure 9). The ability of water lily petioles and leaves to hyperaccumulate Na might be the physiological basis for salt tolerance in water lilies. Water lilies are part of the ANA-grade (Amborellales, Nymphaeales, and Austrobaileyales) angiosperms, and may have retained their genetic potential for salt tolerance from the ancient marine plants that they are descended from.

Materials and Methods
Plants are often subjected to Na toxicity and K deficiency simultaneously. Sodium itself is antagonistic toward K absorption by cells because Na uptake is linked to K efflux [43,44]. Maintaining intracellular K homeostasis is important for proper plant growth and salt tolerance [45]. Our results demonstrated that the potassium content in the leaves and petioles of water lilies decreased after salt stress, with most of the decline occurring within three days of treatment. After three days, the K content stabilized. It appears that K efflux occurs as an early response to salt stress before the petioles and leaves adapt to prevent further loss of potassium ( Figure 9). We hypothesized that the intracellular K concentration may be regulated by potassium-related transporters. Both upregulation (Cluster-3509.83265 and Cluster-3509.8544) and downregulation (Cluster-3509.60348 and Cluster-3509.61628) of potassium transporter genes were observed in the petioles ( Table 2). Water lilies may achieve K homeostasis in the petioles to improve salt tolerance. However, the leaves may use a different strategy to prevent K accumulation. Our results showed that three potassium transporter genes were downregulated in the leaves, suggesting that potassium channels may close under salt stress. ABC transporters are also involved in ion channel regulation under salt stress in plants [46][47][48]. In A. thaliana, a high expression of AtMRP5 led to an increase in the ratio of Na to K in seedlings by regulating K uptake in roots to counteract salt stress [49]. In this study, 27 ABC transporter genes were differentially expressed under salt stress. Most of these transporters belong to the ABCC and ABCG subfamilies, which play an important role in Na and K uptake and transport under salt stress [50][51][52]. Our results suggest that water lilies maintain intracellular K above a minimal threshold to acquire salt tolerance.
In summary, floating leaves and submerged petioles use a series of adaptive strategies to survive salt stress. The absorption and transport of ions and nutrients from the surrounding environment are part of this complex biological process. The ability to hyperaccumulate Na may be the physiological basis for salt tolerance in this aquatic plant.

Plant Materials and NaCl Treatment
The water lily cultivar N. 'Colorado' was obtained from the Nanjing Yileen Garden company. Plants were cultivated in the greenhouse at the Institute of Botany in Jiangsu province and the Chinese Academy of Science. Each tuber was grown in a pot with pond sludge. Then, potted water lily plants were cultivated in a pond. Plants with 5-6 floating leaves were used as experimental materials. For the control plants, potted water lily plants were transferred into deionized water. For salt stress, plants were treated with 50, 100, 150, and 200 mM NaCl, respectively.

Plant Biomass Measurements
After a month-long salt stress treatment, images of water lily plants were taken using a camera (Canon, Melville, NY, USA, EOS 70D). Every floating leaf was scanned using a ScanMaker i800 plus (MICROTEK, Atlanta, GA, USA) scanner. Adobe Photoshop 2022 was used to calculate the leaf area. The fresh weight of the petioles and floating leaves was measured using an electronic balance (Sartorius, Göttingen, Germany, BSA223S). The samples were dried in an oven at 105 • C for 30 min and then dried at 72 • C until no more weight was lost, to measure the dry weight.

Measurement of Gas Exchange and Chlorophyll Fluorescence Parameters
A Li−6800 Portable Photosynthesis System (LICOR Inc., Lincoln, NE, USA) was used to determine gas exchange parameters and chlorophyll fluorescence parameters from 8:00 am to 11:00 am [53]. Each treatment contained ten biological replicates. The concentration of carbon dioxide used in the measurement was 400 mg/L, the light intensity was 1200 µmol/(m 2 ·s), and the measurement area was 2 cm 2 . Gas exchange parameters included: net photosynthetic rate (Pn), intercellular CO 2 concentration (Ci), transpiration rate (Tr), and stomatal conductance (Gs).
After 30 min of dark adaptation, the minimum fluorescence Fo was recorded. A rectangular flash of 8000 µmol/(m 2 ·s) fluorescence was used for excitation and the maximum fluorescence Fm was recorded from dark adapted plants. Actinic light of 1200 µmol/(m 2 ·s) was used to record Fm and steady-state fluorescence Fs under light adaptation. Chlorophyll fluorescence parameters, including maximum quantum efficiency of photosystem PSII photochemistry (F v /F m ), PSII actual photochemical quantum yield (Φ PSII ), electron transport rate (ETR), and photochemical quenching coefficient (qP) were calculated.

Transcriptome Analysis of Petioles and Floating Leaves
The leaves and petioles treated with 150 mM NaCl for 3 weeks were collected for RNAseq analysis. Water lilies raised in deionized water were included as a control. Each treatment had three biological replicates. After collection, the samples were immediately frozen in liquid nitrogen and stored at −80 • C. The total RNA was isolated using an RNAprep Pure Plant Kit (Tiangen, Beijing, China) according to the manufacturer's instructions.
Purified mRNA was used for library construction. The qualified libraries were sequenced using an Illumina NovaSeq 6000 system. Raw reads in fastq format were cleaned using in-house Perl scripts. Raw data were filtered using fastp (Version 0.19.7). Clean reads were obtained by removing reads containing adapters, N bases and low-quality reads. Q20, Q30, and GC content were calculated. The clean reads were assembled into transcripts using Trinity software (v2.4.0). The transcripts were matched to the NCBI RefSeq database. Gene function was annotated based on Nr, Nt, Pfam, KOG/COG, Swiss-Prot, KO, and GO. FPKM (Fragments Per Kilobase of transcript sequence per Million base pairs) values were calculated to quantify the differentially expressed unigenes. Differential gene expression analysis was performed using the DESeq2 R package (1.20.0). Goseq (1.10.0) and KOBAS (v2.0.12) software were used for GO enrichment analysis and KEGG pathway enrichment analysis of differentially expressed gene sets [54].

ICP-MS Analysis of Sodium (Na) and Potassium (K) Contents
Water lily petioles and floating leaves were sampled at 0, 3, 12, 21, and 30 days after treatment with 150 mM of NaCl. Fresh samples were dried to a constant weight. For ICP-MS analysis, the samples were digested with a mixture of nitric acid and H 2 O 2 and examined for sodium (Na) and potassium (K) contents using an inductively coupled plasma mass spectrometer (ICP-MS; Agilent Technologies Co., Ltd., Santa Clara, CA, USA) [55].

Conclusions
The mechanism by which water lily plants adapt to salt stress requires morphophysiological and transcriptional regulation in the leaf-petiole system ( Figure 10). This includes the rapid regeneration of floating leaves, a decrease in leaf number and area, and photosynthetic adaptation. Plant hormones, especially auxin, appear to be involved in the morphological adaptation to salt stress. Ion binding in the leaf-petiole system was identified as one of the most enriched pathways under salt stress. Specifically, sodium hyperaccumulation, K homeostasis, and a selective bias toward NO 3 − was observed. We found that the leaf-petiole system functions as a Na hyperaccumulator, suggesting that water lilies might have retained their genetic potential for salt tolerance from the ancient marine plants that they are descended from.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The data that support the findings of this study have been deposited into the CNGB Sequence Archive (CNSA) of China National GeneBank DataBase (CNGBdb) with accession number (CNP0003913).

Conflicts of Interest:
The authors declare no conflict of interest.